Project Summary/Abstract The broad, long-term objectives of the proposed project are to elucidate mechanisms responsible for assembly and activity of RNA polymerase II (Pol II) preinitiation complexes (PICs) at promoters. The TATA- binding protein (TBP), a central component of the PIC, can bind to DNA in vitro with exceptional stability. However, essentially all of the TBP in yeast cells is highly dynamic, with residence times on chromatin of just a few seconds. Prevailing models derived from in vitro observations posit that a stable promoter-bound complex facilitates transcription reinitiation and thereby perpetuates the activated state. In contrast, we find that dynamic turnover is critical for global transcriptional control in vivo. TBP's dynamic behavior is a consequence of the activity of Mot1, an essential, conserved, Snf2/Swi2-related ATPase that uses the energy of ATP hydrolysis to dissociate TBP from DNA. The goals of this proposal are to use Mot1 as a model to better understand how members of this critical class of enzymes use ATP hydrolysis to disrupt protein-DNA interactions, and to understand why Mot1-mediated dynamics are essential for transcription in vivo. In the first Aim, the Mot1 biochemical system and recent structural data will be exploited to test a specific model for the Mot1 catalytic mechanism. Emerging evidence indicates that cell-to-cell gene expression variation (noise) impacts a wide range of cellular responses to stress and developmental cues, but mechanisms regulating noise are not well understood. Preliminary results show that Mot1 has a previously unappreciated role in suppressing transcriptional noise across a cell population. In Aim 2, we will define the scope and function of Mot1 and other related global regulators in establishing noise levels, and correlate this variation with PIC dynamics. In Aim 3, we will define a newly discovered functional relationship between Mot1 and FACT, an essential regulator of chromatin structure and dynamics. In addition to determining how Mot1 and FACT work together in vivo, new functions of Mot1 in chromatin structure regulation will be addressed, including the unexpected ability of Mot1 to bind to nucleosomes and to regulate the interaction of FACT with nucleosomes. Precise transcriptional control is essential for normal cell growth and development, and numerous transcriptional defects have been linked to human diseases including cancer. Defects in human Snf2/Swi2- related protein complexes are known contributors to certain cancers, Cockayne's Syndrome, alpha- thalassemia, and the most common form of X-linked mental retardation. PUBLIC HEALTH RELEVANCE: Project Narrative The information encoded in the genome is accessed by the process called transcription. Appropriate transcription is critical for normal human health and defects in transcription are known to underlie many human diseases including cancer. The goal of this proposal is to understand fundamental mechanisms controlling the assembly and activity of the cellular transcription machinery.